Respiratory viral co‐infections in patients with COVID‐19 and associated outcomes: A systematic review and meta‐analysis

Abstract The aim of this systematic review and meta‐analysis was to critically assess the published literature related to community‐acquired viral co‐infections and COVID‐19 and to evaluate the prevalence, most identified co‐pathogens, and relevant risk factors. Furthermore, we aimed to examine the clinical features and outcomes of co‐infected compared to mono‐infected COVID‐19 patients. We systematically searched PubMed, Web of Science, Embase, Scopus, and The Cochrane Library for studies published from 1 November 2019 to 13 August 2021. We included patients of all ages and any COVID‐19 severity who were screened for respiratory viral co‐infection within 48 h of COVID‐19 diagnosis. The main outcome was the proportion of patients with a respiratory viral co‐infection. The systematic review was registered to PROSPERO (CRD42021272235). Out of 6053 initially retrieved studies, 59 studies with a total of 16,643 SARS‐CoV‐2 positive patients were included. The global pooled prevalence was 5.01% (95% CI 3.34%–7.27%; I 2 = 95%) based on a random‐effects model, with Influenza Viruses (1.54%) and Enteroviruses (1.32%) being the most prevalent pathogens. Subgroup analyses showed that co‐infection was significantly higher in paediatric (9.39%) than adult (3.51%) patients (p‐value = 0.02). Furthermore, co‐infected patients were more likely to be dyspnoeic and the odds of fatality (OR = 1.66) were increased. Although a relatively low proportion of COVID‐19 patients have a respiratory viral co‐infection, our findings show that multiplex viral panel testing may be advisable in patients with compatible symptoms. Indeed, respiratory virus co‐infections may be associated with adverse clinical outcomes and therefore have therapeutic and prognostic implications.


| INTRODUCTION
In December 2019 a novel virus, severe acute respiratory coronavirus 2 (SARS-CoV-2) was first described in Wuhan, China. Since then, the enveloped RNA betacoronavirus has spread across the world and is currently associated a global pandemic as declared by the World Health Organization (WHO) in March 2020. 1 Since the onset of the pandemic, an immense number of studies has been performed to comprehend the disease and its pathophysiology as well as to generate new therapeutic approaches. 2 Co-occurrence of respiratory infections may be one of the factors that leads to an increased disease severity. 3 Due to their mode of transmission, which is mainly by droplets, respiratory virus circulation within the community is high and dual infections within their realm are widespread. In the pre-COVID-19 pandemic era a 10th of respiratory virus infections was found to be co-infected with another respiratory virus. 4 Various studies observed an increased severity of disease in co-infected patients, especially elderly and high-risk patients. 5 While several studies have reported the co-detection of SARS-CoV-2 with additional respiratory viruses, questions remain regarding the clinical relevance. Virus-virus interaction (either direct or immune-mediated) can have effects on disease severity, transmissibility, immunopathology, and vaccine effectiveness. With SARS-CoV-2, including its variants, becoming firmly established in the human population, it is important to investigate the possible consequences of respiratory viral co-infections (RVCI). 6,7 Data on the prevalence and the most common co-infecting viruses will help clinicians to implement appropriate infection control measures and treat patients adequately, including administering an adequate antiviral therapy whenever available and appropriate.
Knowledge on risk factors for co-infection and on the possible changes in terms of clinical progression of the disease is important to assess the patients' prognosis.
The aim of the present systematic review was to critically assess the published literature related to community-acquired viral coinfections and COVID-19 and to evaluate the prevalence, most identified co-pathogens, and relevant risk factors. Furthermore, we aimed to examine the clinical features and outcomes of RVCI compared to mono-infected COVID-19 patients.

| METHODS
This systematic review followed the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) guidelines 8 (Table S6) and was registered to PROSPERO (registration number: CRD42021272235).

| Search strategy and selection criteria
PubMed, Web of Science, Embase, Scopus, and The Cochrane Library were systematically searched for studies published between 1 November 2019 and 13 August 2021 in the English or German language. Search terms were combinations of three concepts comprising of COVID-19 related words (e.g., 'SARS-CoV-2, '2019 nCoV'), coinfection related terms (e.g., 'concurrent infection', 'dual infection'), and the names and its variants of each of the 18 included respiratory viral co-pathogens. A complete description of our search strings is available in Table S1. The inclusion criteria for the studies included (i) COVID-19 diagnosis according to the WHO COVID-19 case definition 9 ; (ii) patients of any age, setting and severity of illness; (iii) a test for co-infection with any of 18 predefined respiratory viruses within 48 h of COVID-19 diagnosis. Publications that were excluded included: (i) case reports and case series with less than 10 participants, (ii) reviews, (iii) conference abstracts and (iv) studies thematically unrelated to the study objective (Table S2). independently extracted data from the individual studies using a predefined template. Discrepancies were resolved by discussion between the two reviewers. The extracted data included information regarding the study itself (authors, publication year, study design, location and setting, period of investigation, study population) as well as the proportion of co-infected patients and the pathogens implicated, method and time of detection of co-infection, and characteristics describing the mono-infected (SARS-CoV-2 only), and coinfected subgroup (gender distribution, ICU-admission-rate, symptoms, case-fatality-rate).

| Nomenclature and definitions
Terms for virus species and standard abbreviations are approved by the International Committee on Taxonomy of Viruses. 10

| Risk of bias assessment
The quality of included studies was examined using the Newcastle-Ottawa Scale (NOS). 12 The tool provides a maximum score of 4 for selection, 2 for comparability, and 3 for outcome. High-quality studies have a score of >7 and moderate-quality studies have a score of 5-7. Quality assessment was performed independently by two authors (HK, LK).

| Outcomes
The main outcome we sought to analyse is the proportion of COVID-19 patients who were co-infected simultaneously with other respiratory viruses and to describe the co-pathogens. Separate prevalence analyses were conducted for subgroups based on patients (gender, age) and study characteristics (location and time of investigation, cohort size of patients recruited). As secondary outcomes of interest, the rates of (i) respiratory tract infection symptoms (cough, fever, dyspnoea), (ii) intensive care unit admission, and (iii) fatality amongst co-infected COVID-19 patients were further assessed and compared to mono-infected COVID-19 patients. In addition to the co-infection associated clinical outcomes, to evaluate the patients' gender as a potential risk factor for co-infection, the rates of co-infection among the male and female patients were compared.

| Data analysis
Statistical analysis was carried out using the R (v4.1.2) package meta weighting. Risk of publication bias (RoB) was assessed using a funnel plot of the logit transformed prevalence and inverse variance and tested using the metabias function with linear regression 14 and rank correlation test 15 for asymmetry. The quality of evidence (QoE) was evaluated using GRADE methods, which cover RoB, inconsistency, indirectness, imprecision, and publication bias. 13 The QoE was evaluated for each outcome and described in the summary of findings tables, which were created with the GRADEpro GDT software. 16

| Characteristics of the included studies
Approximately two thirds of the studies were cohort studies (41/59).  Table S3.
The risk of bias was assessed by the NOS and is presented in Figure S1. The NOS score ranged from 6 to 9, with a median score of 7.3, which is indicative of moderate quality. Twenty-two studies (37%) are rated as having high quality.

| Comparison of SARS-CoV-2 mono-and co-infected patients
The impact of RVCI on the patients' outcome was investigated by comparison of the mono-infected (SARS-CoV-2 only) and co-infected COVID-19 patients (Table 1). While there were no major differences in the occurrence of cough and fever, co-infected COVID-19 patients were more likely to suffer from dyspnoea than SARS-CoV-2 monoinfected patients (48.1% vs. 37.3% of the patients, respectively). Almost equal rates of patients needed to be submitted to an ICU (25.6% vs. 25.3% of the patients), however, the case-fatality-rate was almost three times higher in the co-infected than mono-infected subgroup (18.2% vs. 6.7% of the patients). Patients' gender as a risk factor for RVCI was assessed by exploring the OR of co-infection among the male and female subgroup, which revealed no contributable effect. None of the other secondary outcomes resulted in an OR significantly different from 1.

| Publication bias and quality of evidence assessment
Publication bias was evaluated using funnel plots ( Figure S11).    35 and there is evidence of a much lower co-infection rate (0.01%) for SARS-CoV-1. 36 Our subgroup analyses revealed that the rate of co-infection was significantly higher in paediatric than in adult patients (9.39% vs. 3.51%, p-value = 0.03). These results are consistent with a recent study examining co-infection rates for paediatric (10.0%) and adult (2.4%) patients. 37 The immature immune system in the youngest patients compared to adults and the greater interaction of children can explain the higher probability of co-infection detection. Although meta-analysis revealed a small increase in the co-infection rate for female patients (11.1% in male vs. 15.5% in female, p-value = 0.12), Figure S2-S6). Abbreviations: CI, confidence interval; SARS-CoV-2, Severe acute respiratory syndrome-related coronavirus. a Patients under the age of 18 years. b Size of SARS-CoV-2 positive patients tested for co-infection. c Continents Africa and Oceania are not demonstrated, since only one study was identified for each of them 27,28 . d Summer is not showed, since studies that screened COVID-19 patients for co-infections during the summer months overlap with the Spring or Autumn season. Studies from tropical geographic regions (N = 2 29,30 ) have been left out because they only experience a dry and a wet season. the difference did not reach statistical significance. In the subgroup containing studies with less than 100 COVID-19 patients the prevalence was higher (from 5.01% to 7%). Underpowered studies often contribute little information and possibly cause risk of bias. It is therefore advisable that future reviews should include studies with a higher minimum number of COVID-19 patients tested for coinfections. 38 The prevalences of RVCI differ greatly among continents. This can be attributed to the low number of studies per subgroup, as well as to the different international laboratory infrastructures and temporal course of the pandemic depending on the geographic region.

F I G U R E 3 Meta-analysis of respiratory viral co-infection prevalence among COVID-19 subgroups (
Our study showed that co-infection was associated with a higher case-fatality rate (6.7% in mono-infected vs. 18.2% in co-infected patients), which is consistent with other studies showing a positive T A B L E 1 Comparison of the mono-infected (SARS-CoV-2 only) and co-infected (SARS-CoV-2 and one or more respiratory viruses) patient groups by secondary outcome measurements ( Figure S7-S10)

p-value
Relative effect (95% CI) I-squared association between co-infection and increased risk of death among co-infected COVID-19 patients. 33 Interestingly, while co-infected patients were more likely to be dyspnoeic, fever and cough occurred at similar rates in the mono-and co-infected patients.

| Conclusion
In conclusion, the present systematic review and meta-analysis pro- Fragkou wrote the first draft of the manuscript. All authors had access to all data in the study and have read and approved the published version of the manuscript.